U.S. patent application number 12/308035 was filed with the patent office on 2010-08-19 for make and use of surface molecules of varied densities.
Invention is credited to Xiaolian Gao, Ailing Hong, Peilin Yu, Xiaolin Zhang, Kiaochun Zhou, Qi Zhu.
Application Number | 20100210478 12/308035 |
Document ID | / |
Family ID | 38846588 |
Filed Date | 2010-08-19 |
United States Patent
Application |
20100210478 |
Kind Code |
A1 |
Gao; Xiaolian ; et
al. |
August 19, 2010 |
MAKE AND USE OF SURFACE MOLECULES OF VARIED DENSITIES
Abstract
The present invention relates to quantitative and quantity
aspects of array synthesis and array uses as a device for high
capacity producing synthetic molecules for off-array surface
applications and as an assay device for on-array surface
applications.
Inventors: |
Gao; Xiaolian; (Houston,
TX) ; Zhou; Kiaochun; (Houston, TX) ; Zhang;
Xiaolin; (Sugar Land, TX) ; Hong; Ailing;
(Bellaire, TX) ; Zhu; Qi; (Houston, TX) ;
Yu; Peilin; (Houston, TX) |
Correspondence
Address: |
G. Kenneth Smith
1645 Briarwood Circle
Bethlehem
PA
18015
US
|
Family ID: |
38846588 |
Appl. No.: |
12/308035 |
Filed: |
June 29, 2007 |
PCT Filed: |
June 29, 2007 |
PCT NO: |
PCT/US2007/072609 |
371 Date: |
December 4, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60817498 |
Jun 29, 2006 |
|
|
|
Current U.S.
Class: |
506/18 ; 506/13;
506/30 |
Current CPC
Class: |
B01J 2219/00725
20130101; B01J 2219/00722 20130101; B01J 2219/00659 20130101; C40B
50/14 20130101; B01J 2219/005 20130101; B01J 2219/00675 20130101;
B01J 2219/00527 20130101; B01J 2219/0072 20130101; C40B 40/04
20130101; G01N 33/6803 20130101; B01J 2219/00596 20130101; B01J
2219/00626 20130101; B01J 19/0046 20130101; G01N 33/54353 20130101;
B01J 2219/00605 20130101; B82Y 30/00 20130101; B01J 2219/00648
20130101; G01N 33/6842 20130101; B01J 2219/00576 20130101; B01J
2219/00592 20130101; B01J 2219/00677 20130101; B01J 2219/00585
20130101; B01J 2219/00454 20130101; C07B 2200/11 20130101 |
Class at
Publication: |
506/18 ; 506/30;
506/13 |
International
Class: |
C40B 40/10 20060101
C40B040/10; C40B 50/14 20060101 C40B050/14; C40B 40/00 20060101
C40B040/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was in part made under the funding support by
the National institutes of Health (NIH, award ID numbers:
R21CA126209 and R43 GM078941).
Claims
1. A method for functionalizing surface containing two or more
sites comprising: (a) attaching a first set of molecules of
predetermined concentrations to at least one site on surface; (b)
attaching a second set of molecules of predetermined concentrations
to at least one site on surface; (c) repeating steps a and b to
form a surface wherein the predetermined densities of the two or
more sites are different; (d) treating the surface
density-variation sites with first reactive molecule; (e) treating
the surface density-variation sites with a second reactive molecule
such that the functionalized sites of different surface densities
undergo at least two steps of subsequent chemical reactions to
cause the formation of at least two new chemical bonds per reaction
site.
2. The surface of claim 1 comprising: (a) at least two or snore
density-variation sites for each type of molecules; (b) at least
one or more different types of molecules;
3. The method of claim 1 wherein functionalizing reaction comprises
adding reaction reagents which may be a regular building block, a
terminator, and/or a multiplier in a predetermined
concentration.
4. The method of clam 1 wherein functionalizing surface comprises:
(a) irradiating at least a first site with a predetermined
radiation strength to activate at least one first site; (b)
irradiating at least a second site with a predetermined radiation
strength to activate at least one second site; (c) repeating steps
a and b to form a surface wherein the predetermined irradiation
strength causes the densities of activated molecules at the two or
more sites are different;
5. The method of claim 1 wherein the subsequent reaction is: (a)
adding a first monomer to functionalized and activated surface
containing density-variation sites; (b) adding a second monomer to
functionalized and activated surface containing density-variation
sites. (c) forming a molecular array wherein the surface contain at
least two kinds of molecules and each is simultaneously present at
more than one density-variation sites;
6. The arrays of claim 5 provide molecules on density-variation
sites for detection and quantitative analysis of proteins.
7. A method for functionalizing a surface containing an array of
sites comprising: (a) attaching a first set of molecules of
predetermined concentrations to at least one site on surface; (b)
attaching a second set of molecules of predetermined concentrations
to at least one site on surface; (c) repeating steps a and b to
form a surface wherein the predetermined densities of an array of
sites are different; (d) treating the surface density-variation
sites with first reactive molecule; (e) treating the surface
density-variation sites with a second reactive molecule; (f)
repeating steps d and e such that the functionalized sites of
different surface densities undergo at least two steps of
subsequent chemical reactions to cause the formation of at least
two new chemical bonds per array site.
8. The surface of claim 7 comprising: (a) at least an array of
density-variation sites for each type of molecules, (b) at least
one or more different types of molecules;
9. The method of clam 7 wherein functionalizing reaction comprises
adding reaction reagents which may be a regular building block, a
terminator, and/or a multiplier in a predetermined
concentration.
10. The method of clam 7 wherein functionalizing surface comprises:
(a) irradiating at least a first site with a predetermined
radiation strength to activate at least one first site; (b)
irradiating at least a second site with a predetermined radiation
strength to activate at least one second site; (c) repeating steps
a and b to form a surface wherein the predetermined irradiation
strength causes the densities of activated molecules at the array
sites are different;
11. The method of claim 7 wherein the subsequent reaction is
parallel synthesis of an array of molecules comprising: (a) adding
a first monomer to functionalized and activated surface containing
density-variation sites; (b) adding a second monomer to
functionalized and activated surface containing density-variation
sites. (c) repeating steps a and b to form a molecular array
wherein the surface contain at least two kinds of molecules and
each is simultaneously present at more than one density-variation
sites;
12. The arrays of claim 7 provide molecules on density-variation
sites for detection and quantitative analysis of proteins.
13. A method of in situ parallel synthesis of peptide array
containing density-variation sites comprising: (a) having an array
layout file containing predetermined density-variation sites and
peptide sequences at predetermined positions, (b) attaching a first
set of molecules of predetermined concentrations to predetermined
density-variation sites on surface according to the array layout
file; (c) attaching a second set of molecules of predetermined
concentrations to predetermined density-variation sites on surface
according to the array layout file; (d) repeating steps b and c to
form a surface wherein the predetermined density-variation sites
according to step a are created; (e) treating the surface of step d
with first reactive amino acid at predetermined positions according
to step a so that reaction occurs between the first reactive
molecule and the surface functional group; (f) treating the surface
of step e with a second reactive amino acid at predetermined
positions according to step a so that the reaction occurs between
the second reactive molecule and the surface functional group; (g)
repeating the treatment steps with reactive amino acids at
predetermined positions according to step a to form the peptide
array with two or more peptides located in different
density-variation sites;
14. The peptide array of claim 13 has an array layout containing
density-variation sites equivalent to those of four or more 96-well
titer plates.
15. The surface reaction sites claims 13 contain either protected
amino groups or free amino groups.
16. The molecule of claim 13 which increases the density at an
array site is a side-chain protected lysine.
17. The molecule of claim 13 which decreases the density at an
array site is an alpha-amino protected amino acid and the
protection group is stable under the subsequent synthesis
conditions.
18. The reactive molecule of claim 13 is protected amino acid for
synthesis of peptides.
19. The surface of claim 13 wherein the density-variation sites are
made by in situ parallel synthesis.
20. The surface of claim 13 wherein the peptides are made by in
situ parallel synthesis.
21. The peptide array of claim 13 provides molecules attached to
the surface with sequentially increasing or decreasing
densities.
22. The peptide array of claim 13 containing peptides which have
known binding properties.
23. The peptide array of claim 13 is for detection of proteins and
quantitative analysis of peptide-protein interactions.
24. The peptide array of claim 13 is for measurement of protein
kinase activities.
25. The peptide array of claim 13 is for measurement of protease
activities.
26. The peptide array of claim 13 is for measurement of
phosphoprotein-binding protein.
27. The peptide array of claim 13 is for measurement of antibody
binding.
28. The peptide array of claim 13 is for measurement of protein
binding.
29. The peptide array of claim 13 is for measurement of nucleic
acid binding.
30. The peptide array of claim 13 is for measurement of
polysaccharide binding.
31. The peptide array of claims 13 is for measurement of the
binding or activities of a biopolymer or a conjugate of biopolymer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to the filing date of U.S.
Provisional Patent Application Ser. No. 60/817,498 filed on Jun.
29, 2006; the disclosure of which application is herein
incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to quantitative and quantity
aspects of array synthesis and array uses as a device for high
capacity producing synthetic molecules for off-array surface
applications and as an assay device for on-array surface
applications.
[0005] The present invention specifically relates to methods for
controlling surface molecular densities of an array at different
array sites to create a density-variation array. These arrays will
be applied to on-surface or off-surface applications, such as high
throughput quantitative measurements for protein bindings, nucleic
acid bindings, and sensitive biomarker detections. Arrays
containing density-variation sites are expected to be used for
obtaining binding and dissociation curves of molecular complexes,
and to be especially valuable for clinical and medical applications
where accuracy of the detection and measurements are more demanding
and significant.
[0006] The present invention also relates to the field of modified
array surface for parallel array synthesis to increase the amount
of the molecules synthesized by a factor of at least 10. These high
capacity arrays will be of use for on- and off-surface applications
in the areas of genome-wide and proteome-wide assays. The molecular
modifications also provide bulk material supplies for preparation
of nanoarrays, single molecule arrays, structural studies or
sequence analysis of nucleic acids, proteins, polysaccharides,
other types of biopolymers and chimeric biopolymers, affinity
purification, drug screening, biomarker detection, genetic
analysis, profiling of nucleic acids, proteins or other types of
biomolecules, clinical diagnosis and patient sample analysis, and
forming sequence libraries or bead-based sequence libraries of
nucleic acids, proteins, peptides, or other types of molecular
libraries.
[0007] The present invention also relates to methods for synthesis
of array molecules containing modifications. The array of molecules
synthesized will be used for on and off-surface applications in the
areas of genome-wide and proteome-wide assays. The molecular
modifications provide unique ligand arrays for analytes of not only
proteins, nucleic acids, carbohydrates, but also cell or cellular
organisms. The molecular modifications also provide bulk material
supplies for preparation of nanoarrays, single molecule arrays,
structural studies or sequence analysis of nucleic acids, proteins,
polysaccharides, other types of biopolymers and chimeric
biopolymers, affinity purification, drug screening, biomarker
detection, genetic analysis, profiling of nucleic acids, proteins
or other types of biomolecules, clinical diagnosis and patient
sample analysis, and forming sequence libraries or bead-based
sequence libraries of nucleic acids, proteins, peptides, or other
types of molecular libraries.
[0008] 2. Description of Related Art
[0009] The rapid development of the genomics (genomics, broadly
represents herein the subject areas related to cellular molecules
and their interactions, including nucleic acids, proteins,
peptides, carbohydrates, lipids, metabolic molecules, and other
functional molecules) and related sciences have constantly created
need of miniaturization technologies. It is well known that each
round of miniaturization of an existing technology requires major
advancement in methods and devices. Today, the most talked human
genome of three billion bases and more than 100k proteins and their
isomers is a clear example of the needs. For this large number of
genetic codes and the products of the genetic codes, there have
been mounting information to show that their analysis (finding out
what are the codes) and profiling (measuring how many and how much
of them are present) are essential for understanding of the basics
of the cellular molecular relationships, identify an individual's
genetic proposition, health condition, disease condition, drug
response, immuno-responsiveness.
[0010] Analysis and profiling of biomolecules require specificity
and sensitivity. While these are now common practices in many forms
of assays, these are a single analysis or reaction at a time. In an
ideal situation, at least one component of the assay has no
competition (or cross reaction possibility). The results thus
obtained are likely to be specific. For instance, in analyzing RNA
using Northern Blotting, a specific probe will be selected and used
to hybridize with the target molecule in the absence of the
competition of other non-specific probes. In another example, a
protein kinase assay kit would contain one protein kinase and one
peptide as the substrate of the enzyme or a protein kinase kit
would contain one protein kinase, one peptide and one inhibitor
peptide for measurements of enzyme activities or inhibitory
effects, respectively. The results of conventional assays tend to
be more reliable due to their simplified experimental conditions.
These experiments do not demand many replicates or complete binding
curves for the specificity of the measurements.
[0011] High throughput assays means simultaneously processing many
different samples and/or a comprehensive assay which will also
simultaneously analyze many samples of a biological system. The
current microwell plates run 96, 384, 1536 assays in parallel in
volumes of microliters to submicroliters. The use of these
microwell plates requires liquid handling robotics, plate loaders,
and large storage spaces. The high throughput assays using
microwell plates will not be practical if the assays are a
genome-wide scale, since there will be high cost and impractical
material consumption problems. For instance, if a population of one
million people were tested for 10,000 important genetic regions,
the total experiment number would be 10 billion If each experiment
consumes 10 microliter reagent/solution, a total of 100,000 liter
of assay solution will be needed. Clearly, even though now we
already have rich information about human genomes, genome
association with diseases, single nucleotide polymorphism (SNP),
DNA methylation, histone modification, DNA
insertion/detection/transposition, and other types of genome
information (see National Center of Bioinformatics, NCBI,
http://www.ncbi.nlm.nih.gov/). only few assays are affordable. It
is therefore a necessary step that the assay volumes are reduced
and the assay devices are miniaturized.
[0012] High throughput assays are also widely used for whole cell
and living cell analysis, such as analysis of apoptosis protein
expression or enzymatic activities in cells, cell-based
phenotype-based drug screening, cyto-toxicity, and cell cycles.
Current platforms, such as Beckman Coulter's Cell Lab.TM., support
sample cups, micro centrifuge tubes and 24-, 96- or 384-well
plates, or flow cytometry, analyzing more than 100,000 events per
second. It is now possible to simultaneously analyzing multiple
variables and parameters using fluorescent labels in high content
screening assays. These technological advancements and the
availability of modern optical microscopic instruments, such as
confocal and the total internal reflection fluorescence (TIRF)
Microscopy, are powerful tools for deepening of our understanding
of in vivo systems. There are clear needs of improvements over the
current systems. For instance, often even one event in a large
population, such as a thousand or a million, can be of diagnostic
or research significance. It is desirable to enrich the
cells/events that are of major interest and analyze these
cells/events into great details using appropriate instruments or
methods, such as those discussed above.
[0013] Microarrays are reaction and assay devices which were
developed in the last two decades and increasingly used as high
throughput tools. Microarrays have dimensions in microns for each
array site and a capacity of thousands to one million assay sites
on a square centimeter area. Some assays may use picoliters or less
reaction solution per assay site. Microarrays do not use liquid
handling instruments as used for microwell plates and for the first
time to allow genome-wide assays run in high throughput mode with
significant reduction of reaction solutions used.
[0014] While one approach of high throughput as described above is
to run parallel single reaction assays, many multiplex reaction
assays involving a mixture of assay molecules and analytes have
been developed.
[0015] Multiplex assays have been done using combinatorial bead
libraries with each bead containing one type of molecules, such as
peptides, small organic molecules, oligonucleotides, or other types
of assay molecules.sup.1
[0016] A plethora of miniaturizing devices have been described and
a common feature of the technology advancement is to enable assays
using nanoliter or smaller volumes. However, the degree of
difficulties of having diverse contents in a miniaturizing device
increases rapidly as the size of feature reduces. This has been the
barrier for many research and development devices to become
commercial products of low cost, easy to use, and diverse
applications.
[0017] One method of creating contents of assay devices is to
pre-synthesize or prepare a large number of different molecules and
place them at different reaction sites in the assay device such as
cDNA or spotting DNA oligonucleotide arrays. As the number of
assays increases and the amount of materials decreases, the front
costs of the material preparation become unbearable and some times
it is impossible to synthesize or prepare the large number of
different molecules required for the assay. Therefore, the method
is limited to arrays of a few hundreds of features, such as
antibody arrays.sup.2, carbohydrate arrays.sup.3, or small molecule
arrays.sup.4, but for DNA oligonucleotides, the number of feature
are in thousands to less than thirty thousands. For assays of
nanoliter or smaller volumes, robotic instruments face many
technical challenges and they do not provide sufficient precision
for precisely deliver (or spot) the assay materials to the reaction
sites. The instrument which could provide such capabilities will be
prohibitively expensive for routine laboratorial use.
[0018] Alternatively, methods have been developed for in situ
synthesis of high density arrays of molecules.sup.5 6 7 8 9 10
i.e., molecules are directly synthesized on a solid surface by
stepwise additions of monomers or building blocks. These methods
overcome the difficulties for pre-synthesis and have been widely
used for DNA oligonucleotide microarray-based assays (e.g.
Affymetrix's GeneChip.TM., Nimblegen DNA microarray, Agilent's
ink-jet DNA microarray, Atactic Technologies/LC Sciences'
.mu.Paraflo.TM., DNA/RNA microarrays) and peptide microarray-based
assays (JPT SPOT synthesis on nitrocellulose fabric materials,
Atactic Technologies' .mu.Paraflo.TM. peptide microarrays).
[0019] Most high density arrays contain standard DNA
oligonucleotides due to the limitation of the synthesis methods
which require synthesis monomers specially made for photolabile
deprotection-based synthesis.sup.7 9. For any modification on the
standard chemical moieties, a new photolabile group protected
monomer must be prepared, and the corresponding synthesis for
making arrays will need to be optimized. The extensive effort
involved for a new type of sequences and their unknown outcomes
associated with modifications of the standard synthesis make the
photolabile protecting group-based method limited to arrays
containing standard DNA oligonucleotides. Array synthesis using
ink-jet instrument relies on conventional chemistry and the
chemistry per se can allow incorporation of modified residues.
However, such synthesis will require a compatible synthesis
instrument to accommodate additional reagents and these
requirements also make the ink-jet method limited to arrays
containing standard DNA oligonucleotides.
[0020] A .mu.Paraflo array synthesis method.sup.5 6 overcomes the
above limitations. The method is based on conventional chemistries,
such dimethoxytrityl (DMT) nucleophosphoramidite (amidite)
chemistry for DNA and RNA oligonucleotides; tert-butyloxycarbonyl
(Boo) chemistry or 9-Fluorenylmethoxycarbonyl (Fmoc) chemistry for
peptide chemistry, and light-activated by photogenerated reagents,
such as an acid or a base, for synthesis of DNA and RNA
oligonucleotides and peptides.sup.11 12 13 14 15. The method
incorporates modifications into array molecules by directly using
commercially available monomers, and diverse arrays containing
modified residues have been synthesized. The modifications of
oligonucleotides or peptides confer them novel binding properties
or enzymatic activities. Such arrays have important applications in
not only research but also diagnostics, drug screening, disease
management, and other areas.
[0021] There is a great need in the fields of genomics and
proteomics where assays should be performed in a number of millions
on a nanoliter or smaller volumes, for parallel synthesis of
molecules on smaller scales than the current 96-well plate
synthesis (Illumina/Invitrogen). It is also highly desirable for a
technology to enable the synthesis of diverse molecules, such as
modified oligonucleotides, modified peptides, molecules with tags,
on a high density array synthesis device for the various detection,
quantitation, and lead-compound development off-array surface
applications. In the last decade, array as a format of high
throughput bioassays has been well-accepted but most of the in situ
parallel synthesis array methods (GeneChip.RTM. of Affymetrix,
Nimblegen, Febit, Agilent, Combimatrix) are limited to making
standard DNA oligonucleotides without modifications. Arrays made
for bioassays do not provide the synthesis quality or a means
required for obtaining sufficient quantities and removing the
molecules synthesized from the array surface for use in the
subsequent reactions, such as making nanoarrays, single molecule
arrays, genome-wide sequence specific primers for genomic DNA
amplifications, or genome-wide target specific probes for DNA or
RNA sequences. Recently, a uPicoArray reactor was described for
synthesis of thousands of DNA oligonucleotides in parallel,
removing the product, and assembling large DNA constructs.sup.11
16. The DNA constructs were protein expression vectors for
generation of proteins of natural types or by design. The DNA
synthesis using miniaturized device reduces the cost by a factor of
at least 30 times and increase throughput by a factor of at least
ten. There is no need for liquid robotic instruments for handling
the samples. Although it is possible to recover each type of
molecules synthesized individually, many applications of multiplex
reactions require molecular libraries and thus a large number of
different molecules are used as a mixture or subgroups of
mixtures.
[0022] In the area of diversify synthesis of array molecules, there
have been great interests in glycol-conjugates, such as
glycopeptides, and glycan analogs due to their important roles in
understanding carbohydrate mediated cellular activities, cell
migration, cell-cell interactions, cell-protein interactions,
protein-protein interactions.sup.3. These interactions are
implicated in a number of pathogenic, immunogenic pathways that are
responsible for infectious diseases, ill-elicitation of
immunoreponses, fertilization, embryogenesis, lymphocyte
trafficking, tumor genesis, and cancer metastasis. Therefore,
glycosylated peptides are of the potential as vaccine, therapeutic,
and model compounds. To date, glycopeptide-based assays rely on
traditional immunological methods at low throughput, but there are
a large number of structural isomers of these compounds. Our
understanding of this complex group of compounds is rather limited.
The synthesis of arrays of glycopeptides and glycosyl modified
peptides can accelerate the studies in this important area and
development of glycopeptide-based agents for assay, diagnosis, and
therapeutic applications.
[0023] One distinct advantage of assays using an addressable array
format compared to a random array is that there are tractable assay
sites as references, positive or negative controls, and other types
of controls and replicates of data points for data processing and
analysis. These are necessary features for ensuring high quality
and reliable assay results. Performing functions such as quality
control analysis of synthesis or assays, baseline signal
correction, normalization of the signal intensities within the
array or of different data sets for generation of reproducible
results and deriving quantitative results are depending on these
features. On addressable arrays, it is always possible to design
and test comparison data sets to exclude the possibilities of false
positive and/or negative signals. The synthetic bead molecular
libraries without a decode mechanism cannot meet these
requirements.
SUMMARY OF THE INVENTION
[0024] Described herein are methods for controlling the molecular
densities of the each of the surface reaction sites in in situ
parallel synthesis of an array of molecules. These
density-variation molecular arrays will find applications in assays
which are normally performed in microtiter plates (such as 96-well,
384-well microtiter plates) loaded with assay molecules or
concentration-variation substrates or assay molecules, detection
and profiling of biomolecules such as nucleic acids, proteins,
antibodies, enzymes, glycans, and a variety of biomolecules.
diagnostic assays, disease treatment and personnel health care
analysis, and other miniaturized bioanalytical assays. These
density-variation molecular arrays will also find applications in
assays of biomolecular complexes, cellular components, cell
membrane complexes, and whole cells.
[0025] The density-variation features will be controlled by mixing
of reagents of those which are regular synthesis monomers that can
react with the functional groups on surface and allow subsequent
deprotection and monomer coupling reactions and those which can
react with the functional groups on surface but do not allow
subsequent monomer coupling reaction (terminators). The ratio of
the mixture of the reagents determines the dilution factor of the
surface density.
[0026] The density-variation features will be controlled by using
reagents which can react with the functional groups on surface and
also allow sequential reactions to create additional reaction sites
at a ratio larger than one (multipliers).
[0027] The density-variation features will be controlled by surface
modifications with immobilized beads which contain functional
groups and thus allow subsequent reactions.
[0028] The method of immobilizing beads on surfaces for in situ
parallel synthesis will be described. Beads will be of different
sizes, shapes, and/or made from different types of materials
suitable for multistep reactions or syntheses.
[0029] The above methods for controlling density-variations on
surface may be used in combination to create variations in integral
or non-integral number of fold changes
[0030] Described herein are also methods for minimizing the effects
of synthesis quality on offset of data quantities. When diverse
molecules are synthesized on surface, their synthesis quality is
not always known. Therefore, it is necessary to deduce the effect
of the synthesis quality to final data measurements.
[0031] Described herein are also methods of synthesizing molecules
on surface which are modified from what would be obtained from
standard, well-known chemistry of synthesis. One method discloses
modifications using Huisgen cycloaddition reaction (click
chemistry).sup.17
[0032] Described herein are also methods of synthesizing molecules
on surface which are modified from what would be obtained from
standard, well-known chemistry of synthesis. One method discloses
modifications by attaching beads to the molecules synthesized to
form bead conjugated molecules.
[0033] The methods of synthesis described herein are not limited to
one type of molecules per reaction site: two or more different
molecules can be synthesized within one reaction site by using
mixture of building blocks or in two separate synthesis steps.
[0034] The molecules synthesized on surface using at least one of
the methods described above will be either used while attached to
the surface or cleaved from surface and collected as a mixture or
subgroups of mixtures. Cleavage will allow occurring at different
bond linkages of a molecule and will in some cases generate a pool
of bead-attached molecules with one bead containing one type of
molecules.
[0035] The applications of the molecules in forms of on- or
off-surface include but not limited to single molecular arrays,
nano-arrays, bead-arrays, bead-based DNA sequencing, single
molecule sequencing, synthesis purification of the molecules
synthesized, affinity purification of analytes, multiplexing PCR,
genome-wide target specific PCR, genome-wide target specific
binding and assay, construction of oligonucleotides, peptides,
carbohydrates/oligosaccharides or other kinds of molecular
libraries.
BRIEF DESCRIPTION OF THE FIGS. AND DRAWINGS
[0036] FIG. 1 illustrates the concept of density-variation reaction
or assay sites in a molecular array.
[0037] FIG. 2 illustrates a set of density-variation array
sites.
[0038] FIG. 3 is a Scheme for synthesis of surface molecules on
array sites of varied-densities.
[0039] FIG. 4 is a bar-graph of the fluorescent signals of the
epitope peptide YPYDVPDYA synthesized on varied-density array
sites.
[0040] FIG. 5 is an exemplary molecular density-variation array
sites shown as a fluorescent image after directly labeling of the
molecules synthesized.
[0041] FIG. 6 is an exemplary molecular density-variation array
sites shown a bar-graph of the measurements from antibody binding
to the molecules synthesized.
[0042] FIG. 7 illustrates an array of protein kinase substrate
peptides and the detection of phosphorylation by phospho-specific
binding agent.
[0043] FIG. 8 illustrates a protein kinase substrate peptide
array.
[0044] FIG. 9 illustrates a protein kinase substrate peptide array
containing density-variation sites.
[0045] FIG. 10 illustrates a plot of the measurements of protein
phosphorylation of a protein kinase substrate peptide array
containing density-variation array sites.
[0046] FIG. 11 illustrates a plot of the chain-length dependence of
the correction factor for the binding data on a peptide array for
compensation of synthesis deficiency which negatively affect the
binding signal intensities.
[0047] FIG. 12 displays the images of a portion of a microfluidic
array chip. (A) Image has empty microfluidic chambers. (B) image
has the chambers filled with immobilized beads for synthesis.
[0048] FIG. 13 is a microscopy image (white light) of
oligonucleotides synthesized on glass plates containing immobilized
TentaGel beads. The image was taken after the oligonucleotide
nucleotide synthesis reaction cycles.
[0049] FIG. 14 illustrates synthesis on bead-loaded array sites and
bead array sites may be density-variation sites.
[0050] FIG. 15 illustrates the Azide alkyne Huisgen cycloaddition
(click chemistry) reaction.
[0051] FIG. 16 illustrates the synthesis of bioconjugate polymer
arrays using azide alkyne Huisgen cycloaddition (click chemistry)
reaction.
[0052] FIG. 17 displays the image of anti-Gal antibody binding on a
glycosylated peptide array.
[0053] FIG. 18 illustrates synthesis of bead-modified molecular
array.
DETAILED DESCRIPTION OF THE INVENTION
[0054] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Still,
certain elements are defined below for the sake of clarity and ease
of reference.
Definition
[0055] Definitions as used herein, the following terms and phrases
shall have the meanings set forth below. Unless defined otherwise,
all technical and scientific terms used herein have the same
meaning as commonly understood to one of ordinary skill in the
art.
[0056] The singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise.
[0057] The following terms are intended to have the following
general meaning as defined in the content after the "dash line" as
they are used herein:
[0058] Activate--In chemical reaction, this means to reduce the
energy of a chemical reaction to induce it to occur.
[0059] Addressable array--An array on which the identities and
specific locations of the molecules are known.
[0060] An array of molecules--A plurality of mostly different
molecules at different locations (spots) of a surface; the low,
medium, high or very high densities of an array is determined by
both total number of spots on the array and number of spots in a
unit area. Currently, an array of several thousands molecules in a
square centimeter area is high density; very-high or ultra-high
arrays has millions of molecules on a slide (1''.times.3''). Arrays
have different properties which are largely depending on the method
of making the array.
[0061] Analyte--A substance or a compound that is an object of
detection or analysis. An analyte of an array experiments may be a
protein, a protein solution, a biological protein samples such as
cell lysate, a nucleic acid sequences, a nucleic acid solution, a
biological nucleic acid sample such as a genomic DNA or total RNA,
treated for array measurements.
[0062] Array control sites, reference sites--An array contains a
number of sites which are not for sample analysis but for technical
quality assessment, such as synthesis, binding, etc.
[0063] Array layout--A two dimensional map of an addressable array,
with the location and sequence identity written in a file.
[0064] Array sites--discrete surface areas on a surface there are
multiple sites.
[0065] Array, chip--These terms are used interchangeably to refer
to a set of sites sharing a common space, the number of sites is
usually at least two in a row and at least two in a column; the
upper limit of the number of sites per row or per column or per
array is set by the dimension for accommodation of a three atom
molecule; the sites have a unit density of at least nine per square
centimeter and the upper limit of the number of sites per unit area
is set by the dimension for accommodation of a three atom molecule;
the sites of an array may be but need not to be regularly located
on the surface. Each array has a predetermined format, such as
planner glass plate with or without array site boundaries. Each
site on an array also called features. The molecules on an
individual site need not to have the same chemical structures.
Important array properties include: array site geometry, surface,
flow through cell array, chamber array, Array subset /// array in a
restricted area (array in 96-well)
[0066] Assay molecules--Molecules that are used for assays; assay
molecules are used for analysis of an analyte.
[0067] Assay sample--A sample containing analyte to be assayed.
[0068] Bead-array--An array with its content delivered by beads
[0069] Beads, particles, microsphere, nanoparticles,
nanobeads--These terms are used interchangeably to refer to small
objects (in dimensions of um to nm). Bead or beads are often used
in the present description. Beads can be made from, but not limited
to, plastics, ceramics, glass, polystyrene, methylstyrene, acrylic
polymers, paramagnetic materials, thoria sol, carbon graphited,
titanium dioxide, latex or cross-linked dextrans such as Sepharose,
cellulose, nylon, cross-linked micelles and Teflon. Beads may or
may not be spherical, may be elongated, may or may not porous, may
or may not be surface coated, may or may not be contain functional
groups such as NH2 or OH may or may not be coasted on surface, may
or may not be having optical properties, magnetic properties,
affinity purification properties.
[0070] Binding/dissociation curve--Plots of the signal of a binding
event as a function of molecule concentration wherein molecule is
an analyte or an analyte interacting ligand.
[0071] Building block, monomer--The terms are used interchangeably;
it is more often to use "monomer" for nucleophosphoramidites or
protected amino acids
[0072] Carbohydrate, saccharide, oligosaccharide, polysaccharide,
glycan--These terms used interchangeably to refer to the
carbohydrate moieties or molecules.
[0073] Chimera or chimeric molecules--A compound containing at
least two groups, motifs, sequences, or moieties from different
common families of molecules; examples of a chimera or a chimeric
molecule include a chimeric DNA-RNA oligonucleotide, a chimeric
peptide from two different proteins, or a chimeric
carbohydrate-peptide.
[0074] Cleavage, conjugate--To break or form a chemical bond
[0075] Conjugate--A chemical bond formed between to molecular
moieties.
[0076] Dendrimers--molecules which has a branched structure,
especially used in this invention dendrimers have multiple branch
arms which can be functionalized to multiply the number of growing
chain of a synthetic molecule.
[0077] Dimension of a three atom molecule--An example of the
molecule is water (H--O--H), whose wider dimension is 2.75
angstrom.
[0078] DNA array--DNA oligonucleotide array, RNA oligonucleotide
array, DNA/RNA oligonucleotide array, RNA array are used
interchangeably for arrays of nucleic acids and
oligonucleotides
[0079] Functionalized surface, synthesis initiation moieties--A
surface is or can be activated for chemical reactions. Often, the
functional groups are ammo or hydroxyl groups or protect amino or
hydroxyl groups
[0080] Glycosylated peptide array--Modification of peptides by
adding carbohydrates to them.
[0081] High capacity array--Array as a synthesizer that can deliver
more molecules (pmol or more per array site) than a regular array
(synthesizing fmol or less per array site).
[0082] Hybridize, wash, strip--Nucleic acid hybridization
experimental steps, hybridization describes the association process
of two complementary strands.
[0083] Immobilization--Reacting of a molecule with surface and
forming a chemical bond between them.
[0084] In situ parallel synthesis--Simultaneous synthesis from de
novo of many sequences molecular arrays
[0085] Linker, surface linker, spacer--"Linker" and "spacer" are
anchoring groups that anchor or tether two molecular moieties; the
chain length of a linker or spacer is determined by their linear
rotatable bonds; linker also defines the molecular moiety anchoring
the solid support with the first molecule on surface; amino or
hydroxyl silanes are often use as linkers on glass surface;
examples of spacer include, but are not limited to, ethyleneglycol
polymer, alkyl, molecules containing branch side chains,
dendrimers, oligonucleotides, peptides, peptditomimetics.
[0086] Modified nucleotide, modified peptide, modified
carbohydrate--A compound which contains chemical moieties that is
different from or additional to those of their natural
counterparts.
[0087] Multiplex reaction, simplex reaction--assays or reactions
involving more than two participating molecules, such as PCR
reaction with ten templates and 20 primers
[0088] Nanoparticles (nanorods, nanoshells, nanocrystals, colloidal
particulates)
[0089] Neutralizing reagent, Sensitizer, Stabilizer--these are
synthesis agents which neutralize acid or base in reaction
solution, transfer irradiation energy, and trapping radical
species.
[0090] Nucleic acid--a polymeric form of nucleotides, either
ribonucleotides and/or deoxyribonucleotides or a modified form of
either type of nucleotide. The terms should also be understood to
include, as equivalents, analogs of either RNA or DNA made from
nucleotide analogs, and, as applicable to the embodiment being
described, single-stranded (such as sense or antisense) and
double-stranded polynucleotides.
[0091] Oligo--when used in combination with a chemical moiety name,
such as oligonucleotides, oligosaccharides, means a short stretch
(i.e., 2, 3, a few, to several hundreds of residues) of the chain
of the chemical moiety.
[0092] Oligos--An abbreviation for oligonucleotides
[0093] On-array surface, on-surface versus off-array surface,
off-surface application of array molecules--Applications using
array molecules on surface; or use chemical or enzymatic means to
remove molecules on surface and recover them for other applications
such as use as an oligonucleotide mixture for mutagenesis or DNA
synthesis.
[0094] Peptide--Oligomer made from amide linkages of 20 natural
amino acids.
[0095] Peptide array--A surface area containing more than 96
peptide sequences immobilized in a square centimeter area
[0096] PGA-P, PGB-P, photogenerated acid precursor or
phostogenerate base precursor. photogenerated acid (PGA),
photogenerated base (PGB), photolabile group protected
monomer--These are parallel synthesis agents, activated by light
irradiation.
[0097] Poly--when used in combination with a chemical moiety name,
such as polynucleotides, polysaccharides, means a long stretch
(i.e., arbitrarily 50, 51, hundreds, thousands, and longer stretch
of residues) of the chain of the chemical moiety.
[0098] Predetermined--an objective which is planed, arranged before
the experiment
[0099] Protecting group, protection and deprotection--These terms
refer to chemical moieties that can block a functional group in a
molecule; the chemical actions are "protection" and
"deprotection"
[0100] Protein kinase substrate peptide array--An array contains
peptides selected from a list of protein kinase substrate proteins
or a peptide array made for protein kinase assays
[0101] Protein phosphopeptide array--an array contains
phosphorylated peptides and array is used for probing
phosphoprotein binding domain proteins, such as SH2 proteins.
[0102] Radiation strength--The dose of the irradiation, measured as
a function of irradiation time and irradiation power.
[0103] Reaction sites, array sites, assay sites--used
interchangeably describing the nature and activities of sites of an
array
[0104] Regular building block--Oligonucleotide or peptide syntheses
use well established DMT-chemistry or Boc or Fmoc chemistry. These
syntheses use standard monomers, which are referred as regular
building blocks. Regular building block also implies their use in
synthesis cycles typically undergoing deprotection and protein
steps.
[0105] SNP, Chip-on-Chip, CGH--nucleic acid analysis and profiling
related terminologies. SNP: single nucleotide polymorphism
Chip-on-Chip: chromatin immunoprecipitation analyzed by chip
(microarray); CGH: comparative genomics hybridization.
[0106] Solid support--Synthetic media as contrast to gas or
solution phases. Solid support include silicon dioxide, inorganic
materials, organic polymers and co-polymers such as polystyrene
resin or beads, grafted polystyrene-polyethyleneglycol resin or
beads.
[0107] Spotting, printing, stamping, spraying--These terms referrer
to the mode of liquid handling.
[0108] Surface--The side/area facing outwards for a planner plate,
bead, or a solid object. A surface is accessible for synthesis or
assay
[0109] Surface density, dilution, concentration, density-variation
sites--Surface density is measured by number of molecules on a unit
surface area. An increase in surface density is "concentration" and
reverse is "dilution". Array sites which have different surface
densities
[0110] Synthesis cycle, synthesis steps--reaction steps repeated in
oligonucleotide or peptide synthesis. Each synthesis cycles is
comprised of several synthesis steps.
[0111] Tag, tagging--An add-on portion of a molecule, protein, or
tissue or cell samples. There are affinity tags such as flag
peptide for antiflag antibody binding, fluorescence dye tag for
labeling and detection of molecules and for cellular component
visualization.
[0112] Terminator, multiplier--these are molecules which terminate
further reaction when incorporated into the molecule synthesized or
which increase the active site when incorporated into the molecule
synthesized. Terminator may or may not be irreversible. Multipliers
may be dendrimers of di, tri, or more dentate branches.
[0113] TFMSA--Chemical name: trifluoromethanesulfonic acid
[0114] The following description of examples is included to
demonstrate embodiments of the invention. It should be appreciated
by those of skill in the art that the techniques disclosed in the
examples are representative techniques discovered by the inventor
to function well in the practice of the invention. However, those
of skill in the art should, in light of the present disclosure,
appreciate that many changes can be made in the specific
embodiments which are disclosed and still obtain a like or similar
result without departing from the spirit and scope of the
invention.
[0115] Described herein are methods for making and use of an array
device with improved capability and capacity in terms of the
variety and the quantities of the molecules synthesized. An array
comprises a plurality of discrete reaction sites which will be used
for synthesis of molecules such as oligonucleotides, peptides,
carbohydrates, their analogues, or other types of organic
molecules. The synthesized molecules at a reaction site are
distributed at a certain density which is measured by how many
molecules contained in a unit area. In one exemplary of linear
peptides which are separated by 40 angstrom apart, there on average
will be fmol amount of molecules synthesized on a 50.times.50 um 2
array site Each array site will require on average nanoliter or
less assay solution for the experiment. When compared with 96-384-,
or 1,536-well titer plates, the arrays described herein will use at
least 1,000 times less assay solution on a per assay (site) basis.
Since increasingly assays of genome- or proteome-wide are run in
tens of thousands and in hundreds of thousands. these experiments
will consume too large of reagents and solutions to make it
impractical to carry out high throughput, large scale experiments
in many laboratories. There will be thousand liters of assay
solutions used and the cost of purchasing, storing, and disposing
them would be prohibitively high. Therefore, significant reduction
in assay materials will remove the barriers for a wide-spread
application of genome- and proteome-wide experiments. These large
scale experiments will provide invaluable information for
understand human biology and advancing biomedical sciences.
[0116] It would be highly desirable to perform the experiments now
run on 96-, 384-, or 1,536-well titer plates in an array format.
One common application of the microwell titer plates is to place
substrate molecules such as peptides in a series of known,
different concentrations into a set of wells on plate and to which
a protein binding solution and then a detection antibody solution
are added. By reading the detection signals which are often
fluorescence, luminescence or colorimetric signals, the
measurements are obtained for deriving biophysical parameters such
as binding/dissociation constants of protein- or antibody-substrate
systems such as enzyme-linked immunosorbent assay or ELISA,
enzyme-substrate activity detection, time dependence parameters of
the various systems containing proteins, nucleic acids,
carbohydrates, lipids, and/or a variety of small molecules. In
enzymatic assays, the density variation sites containing substrate
molecules interacting with enzymes to give data sets which reflect
product formation as a function of the concentration (density) of
the substrate molecules. The experiments can be recorded at various
time points. These data can be used to derive enzymatic reaction
parameters, such as maximum velocity of the reaction (V.sub.max)
and Michaelis constant (K.sub.M). The basics of the above
description can be found in numerous literature articles and
textbooks such as Physical Chemistry.
[0117] Accordingly, the present invention provides synthesis method
for controlling surface molecule densities. Specifically the
present invention reveals methods for preparing arrays of molecules
of constant and varied surface densities; different types of
surfaces are suitable for the synthesis. The applications of the
synthesis include generating high density titer plates to allow
simultaneously performing small volume assays of nucleic acids,
proteins, and other types of molecules quantitatively which by
convention are performed using larger volumes of solutions in
micro-well plates and sometimes in single reaction tubes.
Quantitative analysis provides multiple data points correlated by
quantitative relationships and thus the format of the analysis
provides more reliable information. The outcome should be suitable
for crucial assays directly related to human health, such as
clinical diagnosis, prognosis, monitoring of patient conditions,
genotyping of individuals, and the applications of the types.
[0118] The synthesis methods of the present invention are based on
solid surface containing a plurality of reaction sites or array
surfaces and these sites are suitable for in situ parallel
synthesis. The surfaces for parallel synthesis may be glass,
silicon dioxide, polymer, such as polystyrene, treated
polypropylene, grafted polystyrene-polyethylene glycol, PDMS
(polydimethylsiloxane) and its modifiers, or other materials known
resistant to chemical reagents, such as methylene chloride or
dimethylformamide. For array synthesis intended for on-array
surface applications, the surface will also not to produce
detection interference, such as absorption or emission background
signals which are sufficiently strong to submerge weak signals of
the analyte signals. The surface may be planar or porous, may or
may not contain coating film patterns which define the array sites;
may or may not contain fabrication patterns which define the array
sites. The surface may contain dividers or barriers so that subsets
of array sites are present which will allow differential treatment
of the subset of array sites during and after synthesis. To each
kind of surfaces, a suitable parallel synthesis strategy will be
selected based on the array synthesis technologies known by those
skilled in the art.
[0119] Described in the present invention, the array synthesis will
be carried out on a bead-loaded surface, wherein bead-loading means
immobilization of beads through chemical bond formation between the
array surface and the bead on which in situ parallel synthesis of
molecular arrays will be carried out.
[0120] In an exemplary embodiment, FIG. 1 illustrates the concept
of an array of molecules at different reaction sites with
controlled varied densities. The first row (100) indicates the
position of the density variation sites and the second row (105)
indicates the density variation factor, i.e., by how many fold with
1 being the original density when no dilution or concentration of
synthesis is used. The size of the factor will be depending on the
reaction reagents used. FIG. 1 shows the factor equals two for each
adjacent sites and a total of 2,048 fold of change in molecular
density for 12 sites. In fact, dilution and concentration can be
used in combination to create continuous ratio variations at
various reaction sites. On an array of 3,849 sites, 40 conventional
microtiter plates of 96-wells will be simultaneously created. This
miniaturized assay device will provide at least 40-times faster
throughput but consumes only a fraction of the reagents and
solutions and requires only a fraction of human resources. High
density arrays have even greater potentials, an array containing
100, 200, 400, 800, 10,000, 100,000 or more conventional microtiter
plates of 96-wells will be possible.
[0121] It is clear to those of skilled in the art that surface
molecular density measures number of molecules in a unit area and
thus is a two dimensional parameter, while molecular concentration
measures molecules in a unit space and thus is a three dimensional
parameter. However, these two parameters are comparable if one
considers the spatial separation distance defined by a center to
center distance between two molecules, the relative surface density
of two surface sites is representative of relative concentrations
of two samples in a container. Therefore, the significance of a
density-variation array is in its applicability for quantitative
measurements of the interactions between analyte and surface
molecules. In the presence of an internal calibration, known
sequence and the related density-variation data points, the array
measurements of the unknown analyte can be used to derive
parameters close to conventional biochemical measurements.
[0122] The molecular density-variation sites may be created by
several methods. One approach is to allow partial activation or
reaction at the surface between the surface factional groups and
the added reactive molecules. In an exemplary embodiment, the
surface functional groups are protected amino groups. When the
protecting group is light labile, a full dose of light irradiation
will completely remove the protecting group, a partial dose of
light will only deprotect a fraction of the surface amino group.
The percentage of the deprotection will be proportional to the
strength of the light dose. Alternatively, the surface protecting
group is acid or base sensitive, such as the acid-labile moieties
of DMT or Boc and the base-labile moiety of Fmoc for the amino
group. The percentage of the deprotection will be proportional to
the strength of the light dose which induces acid or base formation
in the presence of photogenerated acid precursor or photogenerated
base precursor as shown in the work of Pellois et al (2001) and
LeProust et al (2001) (Pellois, J. P. Wang, W. and Gao, X. (2000)
Peptide synthesis based on t-Boc chemistry and solution
photogenerated acids. J. Comb. Chem. 2, 355-360; Leproust, F.,
Zhang, H., Yu, P., Zhou, X., Gao, X (2001) Characterization of
oligodeoxyribonucleotide synthesis on glass plates. Nucleic Acids
Res. 29, 2171-2180). However, the light irradiation controlled
methods cannot be used to increase the surface molecular
density.
[0123] Alternatively, chemical synthesis method may achieve
increasing, no change, or decreasing molecular densities on array
sites by adding appropriate reagents (FIG. 2). In one embodiment of
the present invention, the method is to react a multiplier molecule
(205) with the surface functional group to increase the molecular
density at a predetermined site (200). In theory, the surface
density will increase by a factor of two at each step of coupling
of a bidentate dendrimer and at the end of four cycles of
synthesis, there should be eight times more molecules on the same
surface area (210). In another embodiment of the present invention,
the method is to react a regular monomer (215) with surface
functional group to keep the molecular density at a constant (220).
In yet another embodiment of the present invention, the method is
to react a mixture containing terminator (225) to reduce the
molecular density at a predetermined site (230). The relative
concentrations of the reagents used may be according to FIG. 1,
second row of the table (105) wherein #9 spot is according to FIG.
2 (215), such as a Boo- or Fmoc-protected amino acid, and the
protected amino acid couples to surface amino group and the coupled
amino acid will undergo subsequent reaction in next synthesis
cycle. According to FIG. 1, spots #8 through #1 will be synthesized
in eight steps and a doubler such as N,N-(Boc)2LysCO2H or
N,N-(Fmoc)2LysCO2H or N,N-(NPPOC)2LysCO2H (wherein NPPOC is
2-(2-nitrophenyl)propoxycarbonyl, a photolabile group) will be
incorporated. According to FIG. 1, steps #10 to #12 will be
synthesized in three steps and a mixture of 1:1 regular monomer
such as a protect amino acid, such as Boc-Ala, Fmoc-Ala, or
NPPOC-Ala, and terminator amino acid, such as Ac-Ala, will be
incorporated. The mixture reagent will block 50% of the surface
molecules which couple with the terminator amino acid at each
synthesis step. The sequential steps of preparation of
density-variation sites in the three (density increase, no change,
or decrease) are illustrated in FIG. 3.
[0124] In general the amounts of the molecules of each type, or
several types on different sites, or several types on the same
site, made by in situ synthesis on miniaturized devices, can be
increased by several folds or more. This is not limited to the
methods described above but many coupling and chain length
extension reactions can be used. A bidendate dendrimer will
increase the density of the molecules synthesized by a factor of
two for each cycle of synthesis, similar to that of the exponential
growth of the polymer chain amplification. The amplification factor
for a n-branching point molecule will be n.sup.k, where k is the
number of reaction cycles.
[0125] It is clear to those skilled in the art that the actually
surface density of the density-variation factor tends to be less
than that of theoretical due to deficiency in synthesis yield. The
factors that influence the synthesis on density-variation sites
also include surface packing effect. Especially when surface
molecule density increases, the space that allows molecules
aligning in parallel decreases. To certain degree and for certain
molecular systems, there may be unfavorable steric interactions or
surface energies of positive or negative charge, which would reduce
or inhibit molecular density infinitively increase. One must
consider the less than perfect synthesis yield cost by these and
other unfavorable factors when consider the final surface density
of an array site.
[0126] The density-variation sites may be organized like a 96-well
titer plate and the design of the array layout makes careful
considerations to assign each sequence and each density-variation
site a predetermined position/address on the array surface. The
specific density of an array site or its relative density to other
array sites needs not to be identical by using in situ parallel
synthesis method. On different reaction sites, different
"amplification" factors will be possible by synthesizing different
numbers of the branching dendrimers in the growing chains. This
will generate molecules in different stoichiometric ratios. The
molecules synthesized are not limited to peptides but DNA and RNA
oligonucleotides, carbohydrates, and other categories of polymers
and combinatorial synthesized molecules.
[0127] The method of density-variation array synthesis begins with
the design of the density variation patterns and synthesis steps
followed by organizing the array layout for the sequences to be
synthesized. At least a set of density-variation data for at least
one sequence will be collected from the array. The final array
layout file will be used to direct the synthesis of the array on
synthesis instrument.
[0128] In one exemplary of peptide array synthesis, human influenza
virus hemagglutinin (HA) epitope peptide YPYDVPDYA (SEQ ID NO: 4)
was synthesized at density-variation sites. These peptides at
different array sites were coupled with fluorescence molecule in
the synthesis, giving arise to detection signal which is
proportional to the density of the molecules per varied density
site (FIG. 4). The density varied in synthesis from 16-fold
dilution ( 1/16) to 16-fold concentration (16), to give sites of
1/16, 1/8, 1/4, 1/2, 1, 2, 4, 8, 16 in density. The plot shows the
observed ratio of the two varied density sites for each 2-fold
dilution or concentration. The anti-HA antibody (100 g/mL) in
1.times. PBS buffer was applied to the peptide array surface and
the binding data on the density variation sites revealed that the
observed ratio of the two varied density sites for each 2-fold
dilution or concentration. The sigmoid plot illustrated that
peptides of varied densities are equivalent to concentration
variation sites, producing binding curves. This binding curve
produces binding constant in the order of 10 (-7) M by curve
fitting based on a classic isotherm binding model, consistent with
high affinity antibody binding on the epitope peptide array. The
derived binding constant may be used as a calibration value since
the binding constant of anti-HA antibody to its epitope is known to
be in the order of 10 (-8) M. The discrepancy for the measurements
from an array or a solution will be compensated.
[0129] Further binding experiments illustrates simultaneous
measurements of multiple binding curves on a peptide chip
containing peptide ligand sequences. The binding experiment used
anti HA antibody (Roche) and the concentrations of anti HA
(antibody) varied from 0.1 to 60,000 ng/ml. The binding was in TBS
buffer, pH 6.8, one or more hours at 4.degree. C. The signal
intensities were from a secondary antibody conjugated with a
fluorescence dye (cy3 or cy5) which recognize anti Ha bound to
peptide sequences. Signal intensities acquired at different
scanning gains of a laser scanner were scaled according to a
calibration curve of the instrument. Curve fitting used Origin
(Origin Lab) to produce binding constants.
[0130] In one exemplary wherein the synthesis can be monitored by
directly labeling with a fluorescent molecule as the last step of
synthesis or by a fluorescent dye tagged binding molecules to the
molecules synthesized on surface. FIG. 5 is a fluorescence image of
a peptide array containing antibody binding sequences on
density-variation sites. The synthesis of the density-variation
sites varied from using 16-fold dilution (.times.16) in synthesis
cycle 1 to 16-fold concentration (16.times.) in synthesis cycle 9
to give a series of nine spots for each of the sequences: DYKK
DYKW, DYKA, DYA, and a single amino acid A. The relative densities
of these array sites are shown in fluorescence signal intensity
gradient. The fluorescent signal observed for molecule A.
Specifically, the fluorescence intensities were from binding of
cy3-anti-flag peptide antibody (AFM2) to the peptides on array
surface. Peptide sequences are represented by single letter code of
amino acids (A: alanine, D: aspartic acid, H: histidine, K: lysine,
W: tryptophan, Y: tyrosine) (FIG. 6). The synthesis began with a
surface containing Boc-amino groups; selected sites are deprotected
to give free amino groups which were coupled with a mixture of 1:16
of regular amino acid and chain terminator amino acid. The
deprotection reaction was repeated on a second set of sites to give
free amino groups which were coupled with a mixture of 1:8 of
regular amino acid and chain terminator amino acid. These
deprotection and coupling steps were repeated for the mixture of
1:4 and 1:2 of regular amino acid and chain terminator amino acid.
The subsequent deprotection was performed and coupling used regular
amino acid. The deprotection reaction was repeated again to give
free amino groups which were coupled with N,N'-(Boc)2Lysine. The
deprotection and coupling with N,N'-(Boc)2 were repeated three more
times. This nine steps of synthesis produced molecular sites of
varied densities as shown above. The subsequent peptide synthesis
was carried out as described previously. In one example, the
Boc-group was removed from a predetermined set of varied density
sites to give free amino groups which were subsequently coupled
with Boc-alanine. The repeated synthesis cycles of selective
deprotection at predetermined sites and coupling reactions produced
peptide array containing peptides at density varied sites. The
binding curbe plot (FIG. 6) further shows that peptides of varied
densities are equivalent to concentration variation sites,
producing different binding intensities upon binding to antibody.
Peptide array can be used as binding curve titration plate for
monitoring binding and comparative studies of the antibody binding
affinities to a plurality of epitope peptides.
[0131] Antibodies are known to cross binding to peptides which
induce non-specific binding. This property is not desirable for
antibody as purification tag or as therapeutic agent. Peptide high
density array offers a broad range of search of non-specific
binding of antibody to peptides and provide the sequences of these
peptides further the peptide array offers quantitative measurements
(binding curves) of the interactions. These capacities of peptides
make it a powerful tool for specific antibody and epitope
screening, for reducing false positive or false negative
detections. Using binding curves rather than a single binding data
point is also an effective way to identify biomarkers which will
produce more sensitive and reliable measurements.
[0132] Peptide array containing protein kinase substrate peptides
with density-variation sites can be synthesized as described above
for epitope peptide array. FIG. 7 illustrates a form of protein
kinase assay involving peptide chip and protein kinase enzyme. The
peptide sequences on the chip surface are exposed to enzyme and
enzymatic reaction solution. The peptides which are substrates of
the enzyme are thus phosphorylated. The presence of the phosphate
moiety is detected by a staining dye molecule conjugated with a
chemical moiety which specifically recognizes the phosphate group.
Protein kinase assays have many different forms known to those
skilled in the art and thus the peptide chip-based kinase assays
are not limited to the form shown.
[0133] The present invention provides method for universal dye
staining of phosphate on the sequences of a density-variation
peptide array. Fluorescence mage was obtained applying a
phosphate-specific reagent, cy3-Pro-Q (Invitrogen, CA. USA), to
peptide chip containing pS and pT substrate phosphopeptide
sequences. The observed signals are consistent with the present of
pS or pT-containing peptides (phosphopeptides) and the density
gradient curves are also observed. The intensities may be
calibrated for quantitation of the density of the peptides at
individual sites. The phosphate staining reagent may be other
compounds used in magnetic resonance imaging and else where as
contrast agent in biological samples. This method overcomes the
general problem of pS- and pT-peptide detection, since the
antibodies for these peptides gave poor binding and show sequence
specificity. making their detection difficult. The method of the
present invention may be used for sensitive detection of kinase
activities on peptide array and quantitative analysis of kinase
activities.
[0134] The present invention provides method for parallel synthesis
of phosphopeptide array containing density-variation sites.
According to the chemistry and procedures described in FIG. 1, FIG.
2 and FIG. 3, an array containing density-variation sites is first
designed and the proper array surface is functionalized according
to the surface density-variation layout file. On this surface,
phosphopeptide array will be synthesized using a method available
for parallel synthesis of the peptide array, such as the uParaflo
method as described by Zhou et al, (2004).sup.11. Alternatively,
SPOT method may be used as described by Frank and co-workers.sup.18
19.
[0135] FIG. 8 is a fluorescence image of a protein kinase reaction
on pY-phosphopeptide chip. The experiment used Src kinase, p60c-src
(Invitrogen). The chip included 23 known kinase substrates and
their sequence variants, which vary in length and composition, a
number of position and negative control sequences, and a few
artificial peptide sequences. For each substrate peptide, at the
phosphorylation position, Tyr (Y) in YEEI, for instance, three
sequences are present, which are YEEI, pYEEI and AEEI.
pY-containing sequences are directly synthesized which are
reference sequences for the Y-containing sequences (as the
substrate for kinase reactions) There are also background sites
which contain no peptides. 8-22 redundancies were present. The
peptide sequences shown in the image are YVPM (column 1) and the
YEEIP (column 2) related sequences in double replicates. These
sequences are given on right of the image. The results show that
YEE and YEEI are phosphorylated by the Sic kinase used, and
comparatively, YEEIP is a substrate of lower reactivity, and YVPM
has the lowest reactivity. Kinase reaction conditions and
phosphorylation detection were: 10.times. enzyme storage solution
(0.5 mg/ml). 50 ul reaction buffer, pH7.5 (50 HEPES, 0.1 mM EDTA,
and 0.01% Brij 35, 0.1 mg/ml BSA, 0.1% beta-mercaptoethanol, 0.07
mM ATP, 10 mM MgCl.sub.2), 25.degree. C. for 30 min. The array was
stained by filling the array with 200 ul cy3-Pro-Q solution
(phosphate staining reagent), standing at room temperature for 20
min. The chip was then washed with 2 ml destaining buffer, pH 4.0
(50 mM Na.sub.2CO.sub.3, 20% acetonitrile) as recommended by the
vendor. The image was obtained from a regular microarray scanner
(Axon GenPix 4000B) and shown using inverted intensities after
background subtraction.
[0136] The signal intensities were analyzed using the following
equation:
Relative phosphorylation efficiency: f.sub.P
=(I.sub.Y-I.sub.A)/(I.sub.pY-I.sub.A)
[0137] For a peptide, I is the signal intensity, I.sub.Y is for
Y-containing sequence which undergoes phosphorylation by
kinase,I.sub.pY is for the synthetic phosphopeptide, I.sub.A is for
the sequence containing Y.fwdarw.A substitution. By using the
positive controls (pY-sequence) and negative controls (A-sequence
or other non-phosphorylation amino acids) to derive relative
phosphorylation efficiency, f.sub.p as fraction or percentage, the
kinase reaction results were analyzed quantitatively. The method
takes the advantage of addressable peptide array for reducing false
positive readout and improves reliability of the data analysis.
[0138] A protein kinase substrate peptide array gave a fluorescence
image of a PKA assay shown in FIG. 9. Peptide sequences containing
phosphorylated serine (pS) residues are displayed by positive
signal from cy3-ProQ (phosphate-specific staining agent,
Invitrogen). The sequences for each two columns are: 1. RR-X-SL
(6E0 ID NO: 17-31) 2. RR-X-AL, X (SEQ ID NO: 31-44) is labeled in
the FIG. 9. and N-terminus was acetylated. Two redundant data sets
are presented. The varied densities ranges from 32-fold dilution to
16-fold concentration of the original synthesis density using the
synthesis methods disclosed in this invention. Each peptide
sequence was synthesized on these ten varied density sites. The X-S
containing sequences are potential substrates for PKA, X-A
containing sequences are negative controls, and X-pS containing
sequences (now shown) are positive controls. PKA reaction
conditions: PKA kinase reaction buffer (50 mM Tris, 10 mM
MgCl.sub.2, 2 mM ATP, 0.1% BSA, pH 7.5) 1 mL was flow through the
chip for 20 min. This solution was replaced by 2.500U
cAMP-dependent Protein Kinase (PKA) catalytic subunit (New England
BioLabs) in fresh 50 .mu.L of kinase reaction buffer and reaction
continued at 30.degree. C. for 30 min or longer time. The reaction
was terminated with 4 mL. Milli-Q H.sub.2O washing. Detection: 200
.mu.L Pro-Q staining solution (Invitrogen) was circulated through
the chip for 20 min and followed by 4 mL. Milli-Q H.sub.2O washing.
Finally the peptide chip was washed with 4 mL destaining buffer
(100 mM NaHCO.sub.3/CH.sub.3CO.sub.2H, pH 4.5, 20% acetonitrile) at
room temperature before image scanning.
[0139] The corresponding plots of the above density-variation data
from a PKA reaction for 20 RR-X-SL peptide sequences are shown in
FIG. 10. Each of these sequences was synthesized on varied density
sites (FIG. 9). The plot is the measured fluorescence
signal/reaction time (30 min) versus varied densities in arbitrary
units (AU). Separately, at each density point of a peptide, a plot
of velocity (v) versus time of the reaction is determined. The
initial reaction velocity (v.sub.o) is derived from the slope of
this plot in the region where the function is linear, for each
peptide at each density variation point. A similar plot to above
but velocity is v, produces a curve from which maximum velocity of
the reaction (V.sub.max) and the K.sub.M are determined as
described in textbooks of Biochemistry (e.g. Biochemistry, 5th Ed.
Berg, J. M., Tymoczko, J. L., Stryer, L. (2002) W. H. Freeman and
Company, New York, pp. 200-222). These kinetic parameters are also
derived from the intercepts of the x-(=-1/K.sub.M) and the y-axis
(=1/V.sub.max of the Lineweaver-Burke double reciprocal plot.
[0140] The applications of protein kinase substrate arrays are
related to several areas. The peptide array can be a kinase
detection tool for identify a kinase in a complex biological sample
or identification of a novel protein; the peptide array can also be
of use for profiling of cellular kinases. Due to the central role
of protein kinases, these applications will have significant impact
on not only research but also disease diagnosis, therapeutic target
identification, inhibitor screening, for example.
[0141] The present invention provides a method for improving the
quality of quantitative measurements of array experiments. The
following data processing involves procedures for removing noise
from binding signals. In particular this noise is from the
differences in synthesis qualities. For arrays of diverse molecular
moieties, the measured signals become increasingly inaccurate due
to unknown synthesis qualities and it is impractical to optimize
the synthesis to its perfection. Therefore, it is useful if the
synthesis quality parameter can be considered in array data
analysis. The method according to the present invention is in the
last step synthesis on chip to use 1% fluorescein direct coupling
of the surface functional group in the molecules synthesized, such
as the terminal amino group in peptides. Following the synthesis,
the fluorescein was activated and image was acquired. The
fluorescence signal was I.sub.F and the background signal was
I.sub.bg1. This peptide array contained anti flag peptide epitope
and various peptides (total 414 sequences) of 2- to 12-mer in
length with at least four replicates (#050013). The side chains of
the peptides were then deprotected using the TFMSA protocol for
Boc-chemistry synthesis. The array was washed with water and
binding TBS buffer. The binding experiment used 100 ng/ml anti flag
M2 (AFM2) at 4.degree. C., TBS buffer, pH 7.5, 1 hour. The binding
was detected through cy5-IgG staining (100 ng/ml) at 4.degree. C.,
TBS buffer, pH 7.5, 30 min. The cy5 signal of the antibody binding
was recorded using a microarray scanner (Axon GenPix 4000B). The
data sets contained cy5 signal (I.sub.b), background signal
(I.sub.bg2), and from previous image, I.sub.F. From these data raw
binding intensities for binding were determined by
I.sub.b=I.sub.b-I.sub.bg2 and direct fluorescein labeling
intensities were determined by .DELTA.I.sub.F=I.sub.F-I.sub.bg1.
The ratio R.sub.bF (=I.sub.b/I.sub.F) gives binding molecules per
unit fluorescence reading of the synthesis or this is called
corrected binding data. Next, R.sub.bF and .DELTA.I.sub.b are
balanced in overall intensity and the correction fact is derived by
comparing the two binding intensities (corrected/raw) and these are
plotted above for the 414 sequences in FIG. 11. For each length,
two groups of correction factors are observed: those along the line
1110 and those along the line 1120. The slope of hoe 1110 is
greater than line 1120. The sequences of the line 1110 group are
those of greater correction factors when the sequences of the same
lengths are compared. The greater correction factor in this case is
due to lower synthesis yields in certain amino acids. For each
line, line 1110 or line 1120, the trend is the longer the
sequences, the greater the correction factors. This is consistent
with the length effect in synthesis efficiencies. When correction
factor is less than one, such as those for shorter sequences, the
actual assay intensity is less than without the correction or vise
versa.
[0142] The method of binding signal correction for synthesis
deficiencies has a number of benefits. Normally, one assumes the
higher the binding intensity, the stronger the binding without
taking into consideration of the difference in chain length and/or
sequence compositions. However, in one example of peptide
synthesis, the synthesis yield is not 100% and 20 amino acids give
different synthesis yield as well. These deficiencies in synthesis
may lead to erroneous conclusions in analyzing the binding data For
instance, if the stepwise synthesis yield is 95.0%, a 3-mer
sequences will be 1.6-fold of the 12-mer sequences. When the
binding data is evaluated, this difference in synthesis yield
should be considered. The direct fluorescence labeling provide a
means for quantitation of the amount of the synthesized molecules,
allowing the length effect in synthesis to be deducted. By the same
token, the difference in synthesis yield by different amino acids
is also deducted. The assay data are evaluated based on
signal/signal (molecules on surface).
[0143] One should note that fluorescence or other types of labeling
molecules may affect the subsequent assays due to higher background
signals. This can be overcome by allowing low percentage of labels
or attaching the label through a cleavable linker. After the
signals for synthesis are acquired, the labels are removed without
affecting the subsequent applications. A removable linker between
the sequence synthesized on surface and a label may be base labile,
such as ester linkage or 1,2-diol moiety-containing linkers, or
acid labile, such as those cleaved to generate carboxamide
moieties, reductive cleavable, such as the disulfide linker, or
light labile, or enzymatically cleavable, or as those well-known by
those skilled in the art. It is important to select label and
conditions so that the signal reading for synthesis is linearly
proportional to the amount of sequences on surface.
[0144] The above discussions should be generally applicable to the
signal analysis of the array data from enzymatic reactions,
hybridization, and various array assays. For different array
systems such as peptides, modified peptides, phosphopeptides,
dye-tagged peptides, oligonucleotides, carbohydrates, the ratio
parameters will be different and adjusted method for different
synthesis, for different types of molecules, and for different
direct labeling signal used.
[0145] The methods of the present invention as discussed above for
density-variation array design, array synthesis, array quality
control, and array data analysis are not limited to peptide arrays.
The general procedures and steps are suitable for making and use of
DNA/RNA oligonucleotide arrays, carbohydrate arrays, and a diverse
family of arrays containing chemical modifications of these groups
of molecules.
[0146] The present invention provides methods for effective peptide
array synthesis Parallel synthesis using conventional chemistry
that is activated by photogenerated reagents, such as
photogenerated acids or bases, has been demonstrated for synthesis
of DNA/RNA and peptide arrays, and arrays of modified DNA/RNA and
peptides. In one exemplary wherein peptide array is synthesized by
deprotection using a photogenerated acid precursor, triaryl
sulfonium salt. The deprotection time for removal the N-Boc group
on the terminal chain under this condition is more than 10 minutes,
or more than 12 minutes for more complete deprotection of the amino
group, This long time required for peptide array synthesis is
problematic. The comparison of the deprotection efficiency of
sulfonium salt and iodium salt revealed that the latter took only
less than one minute or less than 30 sec. to completely remove the
Boc-protecting group, representing a significant reduction of the
peptide array synthesis time. The new reagent suitable for peptide
array synthesis becomes more important for density-variation
peptide arrays due to increased synthesis steps are required.
[0147] One experiment compared two sets of deprotection conditions:
Reaction A used iodonium salt in the presence of
2-isopropylthioxanthone in CH.sub.2Cl.sub.2 in the deprotection
step for removal of the acid labile Boc group of amino groups on
surface. Reaction B used a different photogenerated acid precursor.
The deprotection used different irradiation times (from 1 sec to 10
min light irradiation). The fluorescence moiety was coupled to the
peptides on surface after all the deprotection steps were
completed. The darker the spot, the stronger the intensities, the
more effective the deprotection conditions. Reaction A is more
effective in 20 sec to one minute than reaction B in 7 or more
minutes. The deprotection of the Boc-group can be achieved on time
scale of seconds. The peptide arrays synthesized using iodium salt
are validated with standard binding experiments using anti-flag
antibody AFM2 and experiments produced comparable results to the
existing
[0148] Reducing the synthesis cycle time is critical for practical
use of in situ parallel synthesis of peptides. The synthesis cycle
time is a function of synthesis instrument and thus improvement in
instrumentation contributes to shortening of the synthesis time as
well. These include measures of stronger light source (without
sacrificing optical resolution), delivering heat to the chip,
microwave application to the chip synthesis area, and other methods
well-known for acceleration of the rate of organic reactions.
[0149] The present invention provides method for significantly
increasing the capacity of an array as a synthesizer. A high
density array surface herein is defined as a high number of
molecules synthesized per unit area, and thus a high capacity array
is comprised of high density array surfaces. One method is to load
and immobilize porous synthetic media to a support surface such as
a planner glass as used at the present time as the surface of array
synthesis. The porous synthetic media such as polystyrene beads
(Pierce, USA). TentaGel beads (Rapp-Polymere, Germany), and
controlled porous glass beads (CPG or LCAA-CPG, Sigma, USA), can
generate at least 10 times more materials and 100, or 1,000 times
or more can also expected. In one embodiment of the present
invention, 10 um TentaGel beads are loaded into a microfluidic
array chip, which was described in Zhou at al. 2004 (Zhou, X., Cai,
S., Hong, A., Yu, P., Sheng, N., Srivannavit; O., Yong, Q.,
Muranjan, S., Rouilard, J. M., Xia, Y., Zhang, X., Xiang, Q.,
Ganesh, R., Zhu, Q., Makejko, A., Gulari, E., and Gao, X. (2004)
Microfluidic picoarray synthesis of oligodeoxynucleotides and
simultaneously assembling of multiple DNA sequences. Nucleic Acids
Res. 32, 5409-5417). The surface of the chip was derivatized with
an amino propylsilane linker and the amino group was then reacted
with succinnyl anhride. The beads (TentaGel M NH2, 10 urn) were
from Rapp-Polymere (Germany). The beads were loaded into the array
sites (microfluidic chambers) (FIG. 12, where in 1210 has empty
reaction cells and 1215 is filled with the beads). The beads react
with the amino group on array surface and increase the surface
synthesis capacity dramatically. TentaGel beads are wildly used as
solid support for synthesis of oligonucleotides and peptides.
[0150] In an exemplary shown in FIG. 13 wherein oligonucleotides
are synthesized on a glass plate loaded with 10 um TentaGel beads.
The glass surface was derivatized as described by Zhou et al. (2001
(Zhou, X., Cai, S., Hong, A., Yu, P., Sheng, N., Srivannavit, O.,
Yong, Q., Muranjan. S., Rouilard, J. M., Xia, Y., Zhang, X., Xiang,
Q., Ganesh, R., Zhu, Q., Makejko, A,, Gulari, E., and Gao, X.
(2004) Microfluidic picoarray synthesis of oligodeoxynucleotides
and simultaneously assembling of multiple DNA sequences. Nucleic
Acids Res. 32, 5409-5417) and was placed in a 1 umol DNA synthesis
column. The column was connected to a DNA synthesizer (Expedite
8909, Applied Biosystems) and an oligonucleotide 15-mer was
synthesized using the standard DMT chemistry and a standard
synthesis protocol provided by the vendor. After synthesis, the
protecting groups of the nucleotide bases were removed. Four tests
were run to validate the synthesis: (a) direct labeling of the
oligos synthesized by fluorescein and the detection of fluorescence
signals as described in (Leproust, E., Zhang, H., Yu, P., Zhou, X.,
Gao, X. (2001) Characterization of oligodeoxyribonucleotide
synthesis on glass plates Nucleic Acids Res. 29, 2171-2180); (b) UV
absorption scan of the cleaved products from the surface and a
positive reading at 260 nm; the glass plate loaded with TentaGel
beads produced at least 1,000 times more oligos compared to that
produced from the glass surface; (c) HPLC analyzed the oligo
produced on bead-loaded glass plates, the observed retention time
was 16 min, in agreement with a standard sample (HPLC column (RC):
C18, 8.times.10 10.mu.; flow rate: 1.5 mL/min, UV wavelength
monitored: 200-600 nm; solvent A is CH3CN, elution solution B is
0.05M TEAA buffer plus 1% CH3CN, gradient: 5% A 2 min, 5-35% A, 20
min); (d) hybridization on bead-loaded glass plate. The
hybridization complementary strand of the oligo synthesized on
bead-loaded plate was synthesized on CPG support and 500 uL
solution of 0.5 uM hybridization cy3-labeled oligo was used. For
comparison, a bead-loaded glass without synthesis of oligos was
treated by the same procedure as that used for bead-loaded glass
plate with synthesis of oligos. The glass plates were imaged under
epifluorescence microscope and stronger fluorescence signal was
observed from the glass plate containing synthetic oligos.
[0151] In the method of the present invention described above,
oligonucleotides synthesized on the bead-loaded glass plate were
removed from surface for off-array surface applications (FIG. 14).
It is not necessary that all array sites to have the same surface
density or capacity (14100, 14105. 14110). The linkages between the
surface and the beads and between the bead and the oligonucleotide
are stable under synthesis and deprotection conditions of
oligonucleotides as described in (Gao, X. and Yu, P. "Novel
reagents compounds and methods of making and using the same". PCT
International Publication WO 2005/000859). One example of such a
linker is the amide bond linked alkyl chain. For on-array surface
applications (14115), there are a large fold more surface molecules
at each array site; this will lead to more sensitive detection. The
dynamic range of the detection may also increase since while the
low detection limit is about the same as that of conventional DNA
oligonucleotide arrays, the upper detection limit is much higher.
For off-array surface applications (14,120, 14,125), the cleavage
bond is designed so that the cleavage is to occur to release either
products without the bead attached (14,120) or with bead attached
(14,125). Although the cleavage can occur during the final
deprotection stage after the molecule is synthesized, it is
desirable to cleave the product synthesized at the last step
succeeding the final deprotection steps. The subject of linker use
in oligonucleotide synthesis is discussed in detail (Gao. X. and
Yu, P. "Novel reagents compounds and methods of making and using
the same". PCT International Publication WO 2005/000859).
[0152] In the method of the present invention, the bead-loaded
array capacity is determined by the specific configuration of the
loading beads. The example used herein is 10 um TentaGel beads
which have sub-picomol per bead functional groups and there are
millions beads per gram of bead weight. If the array site is of the
size 50.times.50 square micron, there may be up to hundreds of pmol
molecules or more than 10 (14) molecules synthesized per array
site. Therefore, the bead-loaded glass plate as media of synthesis
produces, in one example, wherein nmol of molecules synthesized
(FIG. 13). Importantly, in an array synthesizer, hundreds,
thousands, up to millions of different molecules are simultaneously
synthesized; while it would require many batches of synthesis is
required for the prior art of 95- or 384-well synthesis for making
up to millions of different molecules. The use of chemical
materials by the prior art of synthesis will also be prohibitive
for it to be useful in the very large scale applications. It is
well-appreciated by those skilled in art that array synthesizer has
major advantages over the prior art of micro-well based synthesis
and is essential for nano- or smaller-scale bioassay devices
running ultra-high throughput genome- or proteome-wide
experiments.
[0153] The method demonstrated herein are not limited to the
materials of glass plate and TentaGel beads, not limited to the 10
um beads, not limited to spherical supporting for synthesis. Other
sources of materials of different chemical (such as PDMS,
polystyrene) or physical (such as total plat, having etched,
layered, multi-sided) configurations for high capacity synthesis
appreciated for those well-known in the art may also be choices as
well for intended array synthesis. The surface of the array may be
modified to contain wells, holes, shallow or deep channels, or
other physical modifications so that the surface of the array is
divided into subsets of areas, or add other feature to facilitate
subsequent procedures of bead loading, synthesis, and the on-array
or off-array surface applications. Considerations for beads of
choices are multiple: bead size can vary such as from urn to nm;
bead geometry and morphology may be spherical, hollow (nanoshell),
square, elongated (rods); bead properties include magnetic,
optical, or other types of active functions; bead materials may be
polymers, crafted polymers, gold, silver, or other metallic
composites, nanocrystals, or other materials which can form
functional beads in desirable dimensions. Beads of desirable
properties possess one or a combination of the properties described
above.
[0154] The method demonstrated herein are not limited to
oligonucleotide synthesis and not limited to one type of molecule
within one array site. It is well-appreciated by those skilled in
art that peptides, oligonucleotide analogs, peptide analogs.sup.5
6, or in general polyamide and polyphosphodiester sequences,
carbohydrates.sup.3. Synthesis and medical applications of
oligosaccharides. Nature 446, 1046-1051), and their conjugates.
More than one type of molecules can be made on one array site using
methods well known to those skilled in art such as randomization
synthesis or orthogonal synthesis of two or more different
molecules.
[0155] The method demonstrated herein provide a mixture of
oligonucleotides with (one bead is loaded with one type of
sequences) or without beads attached. The mixture of
oligonucleotides have broad applications: the mixture bead samples
can be used for target-specific applications such as probes for
enrich or select DNA for sequencing for bead-based fast DNA
sequencing such as the recent technologies from 454 Life Sciences
and Roche (www.454.com) and Applied Biosystems
(www.appliedsystems.com)), genome-wide DNA sequencing from
Solexa-Illumina (www.illumina.com), nanoarrays, single molecule
arrays, target specific realtime PCR, genome-wide amplification for
various DNA analysis (SNP, Chip-on-Chip, CGH, methylation mapping),
RNA sequencing and profiling, tags for purification, labeling,
detection, or barcoding, bead arrays for assays of nucleic acids,
proteins, and other types of molecules. These applications requires
one, ten, 100, or 1,000 of the same molecules or the probes on
beads and the bead-attached molecules target the same binding
molecules. An array synthesizer producing fmol (6.0.times.10 8
molecules) will provide materials for 100,000 or more experiments.
An array loaded with beads will provide materials for at least
1,000,000 or more experiments. Oligonucleotide mixtures also
provides materials for synthetic DNA as described by Zhou et al
(2004), which are precursors or building block materials for
functional DNA constructs, RNAs, proteins, biopolymer complexes,
minigenomes, organisms or cells.
[0156] In one embodiment of application, a set of 50,000 gene loci
related to cancer gene activation (average length 1.5 kbp) for
1,000,000 test objects will be sequenced at 3.times. (three reads
per sequence) (total 50,000,000,000 sequence read). This task can
only be deal with by the current large scale sequencing
technologies. However, the known technologies, such as 454 DNA
sequencing using blank beads to randomly catch a sequence for
sequencing. In this process, many beads catch too many sequences
and many beads did not catch any sequences; the portion of the
beads loaded with correct number of sequences follows typical
statistical distribution. Furthermore, the beads loaded with a
single sequence may be redundant, the tendency of which is
depending on the abundance of a particular sequence; the higher the
abundance, the high the chance that sequence will be populated on a
larger number of beads. Therefore, there is a need for
target-specific preparation of sequencing samples. A simple
solution is to provide beads loaded with a single probe that can
hybridize with a predetermined target. This simple solution,
however, require high capacity oligonucleotide synthesis. A set of
probes selecting the 3,000 genes from total RNA is provided so that
at each sequencing run, the sequenced sequences are not by random
choice but by hybridization selection. With this capability, the
efficiency of each run is ensured and full sequence coverage is
ensured or else, completion of the task is not possible. It is this
type of deep sequencing effort that can help taking full advantage
of the genome sequences available and gain insights that would not
be possible if research dwells on low capacity low throughput,
random experiments.
[0157] The present invention provides method for chemical
modification on array surface of the molecules synthesized. The
modifications introduce new properties to the array synthesized
molecules and thus open up new avenues for array applications which
are beyond nucleic acid hybridization or typical peptide-based
assays. One exemplary modification reaction is adding carbohydrate
moiety to peptides through covalent linkages to create new
molecules of modified peptides for screening antibody binding, cell
receptor binding, protein binding, vaccine candidate screen, and
other applications of normally involve carbohydrates and small. One
useful reaction is Huisgen cycloaddition (click chemistry) (FIG.
15), which is to react a terminal alkyne (1505) with an azide
(1510) compound to form a trizole (1515); this reaction conjugates
the R and the R1 group to give an extended chain. The linker moiety
in the azide compound 1520 may be an alkyl, ethylene glyco
oligomer, polyamide, etc. Click chemistry has quickly gained
popularity in last few years, due to the fact that reaction
conditions are easy to accommodate and the compatibility with
aqueous conditions suitable for biomolecular reactions. Described
herein an experiment attaching an alpha-gal trisaccharide azide to
peptides to form bioconjugates on an array surface. In a preferred
embodiment of the synthesis (FIG. 16), a peptide array (1605)
contains Boc protected amino surface group. Selective deprotection
of an array site to free surface amino group (1610) which in turn
couples with propdic acid (X=nothing) (1615) to form a termus
alkynyl (1620). Under click reaction conditions, the azide compound
(1625) wherein R is a linker-linked fluoscein, the azido-dye (1625)
is "clicked" to the terminal alkynyl to form the conjugate
(Z=trizole) (1630). On this array surface the reaction steps of
deprotection, coupling an alkynyl carboxylic acid, followed by
Huisgen cyclioaddition are repeated, and a library of bioconjugates
(1635), containing group R1, R2 or R3 at different array sites. In
one example, the bioconjugates are peptide-derived compounds.
[0158] The modification of the molecules synthesized on an array is
not limited to using click chemistry, other bioconjugation
chemistry, such as cross-links formed by maleimide/thiol,
aldehyde/amine or hydrazine, thiol/thiol, and the bis-aryl
hydrazone bond (SoluLink, USA) are among the choices,
[0159] In one preferred embodiment of the present invention, the
reaction involved first synthesize the peptide sequences using
photogenerated acids and Boc-chemistry as described previously.
After the peptides were made, a first set of reaction sites were
deprotected, and the amino groups were coupled with propiolic acid;
a second set of sites were deprotected, and the amino groups were
coupled with 4-pentynoic acid; the steps of deprotection and
coupling were repeated to produce sites coupled with 6-heptynoic
acid. These sites contain terminal alkyne groups. The peptide chip
surface was then reacted with FITC-azide in ethanol and water
mixture and in the presence of CuSO.sub.4 and Ph.sub.3P for 2
hours. A layout of the displayed image in FIG. 17 is provided at
top, a fluorescence image of alkynyl terminated peptide sequences
linked to FITC (fluoroscein isothiocyanate) after the cycloaddition
of FITC-azide and the surface alkynes. The displayed signals are
consistent with the designed layout pattern, demonstrating the
Huisgen cycloaddition works well on surface in array synthesis for
modification of peptides.
[0160] In another preferred embodiment of modification, the
reaction conditions are similar to what described above and the
selected deprotection step and coupling steps are arranged as such
that the modifications are multiple at different positions of a
sequence (1640) (FIG. 16).
[0161] The arrays containing modification molecules such as
glycosylated peptides are of value as biomarker binding ligands due
to their presentation of more functional groups compared to regular
DNA or peptide molecules.
[0162] The present invention provides method of forming modified
molecular arrays using in situ parallel synthesis methods based on
activation of the synthesis cycles by direct photolithographic
light irradiation, using photogenerated reagents of acid or base,
electronically generated acid or base, or moving the reaction agent
to the reaction site. The array of synthesis in not limited to
certain sets of specific configurations, such as arrays of enclosed
devices, or open surface, arrays in subsets or restricted areas,
arrays of square centimeters or smaller sizes. However, it is
critical that an array should allow modification reactions in a
first predetermined area and then in a second predetermined area
and so on.
[0163] The modifications and bioconjugation reactions on array
surface are not limited to glycosyl conjugated peptides,
oligonucleotides, carbohydrates, molecular moieties, these can be
proteins, antibodies, cells, such as TentaGel beads. Beads said
herein also include colloidal CdSe--ZnS or other types of quantum
dots found useful for tissue and in vivo staining, magnetic beads,
coated magnetic beads found useful for quick purification of
samples. Bead surface may be derivatized with functional groups
such as hydroxyl OH, amino NH2, sulfuhhydryl SH, biotinyl,
N-succinimidyl (NHS), azide, alkenyl, or alkynyl. Alternatively,
bead surfaces may be coated with active molecules such as
strepavidin, antibody, GST (glutathione S-transferase), HIS6
(HHHHHH), etc.
[0164] In a preferred embodiment of the present invention, one bead
one compound of a total of Ni thousands to millions of beads
containing different compounds will be in great demand in next few
years for high throughput assays and biochemical applications in a
miniaturized format. These beads may serve as probes for specific
binding, substrates for enzymatic assays, primers for polymer chain
extension, and numerous applications have been used daily now in a
macroscale format. One can envision to reduce the size of the
current applications by using millions of beads of nm on a surface
to form an array. This surface may or may not have grids to
separate the beads. This surface may have coating to retain the
beads. There are a large number of different types of beads made
from different materials, such as gold, synthetic polymers and
grafted synthetic polymers, sol-gel, and glass, and sizes ranging
from nm to um.
[0165] The present invention provides method for preparing a bead
library using array as a synthesis tool. Previous art of the
well-known split-and-pool method produces a bead pool, but this
method is laborious and the random arrays make difficult for
well-controlled studies. Robotic spotting or other methods of
immobilizing molecules onto the beads have been used but these
methods are only practical for a bead pool of thousands of
different compounds This is because the compounds are
pre-synthesized and it is cost- and time-prohibitive to
post-synthesize a larger number of compounds than a few thousands.
The present invention provides method for creating a bead pool
using array compounds synthesized on surface.
[0166] In particular, method for preparation of a bead pool of
directly "copy" the chemical content of a surface containing a
variety of compounds is revealed. A few examples of the disclosed
method are given below.
[0167] The synthesis of the array is performed as described in the
publications.sup.5. For each of the biopolymers synthesized, an
anchor moiety is incorporated, for instance, at the terminus
position. Anchor molecules are those which can form covalent bond
or high affinity bonding with incoming molecules. After the array
synthesis, beads, to which the surface molecules can adhere, are
loaded onto the surface. The surface molecules will bind to the
beads upon contacting. The array surface molecules are then cleaved
from the surface. At each contact area, only one type of molecules
is bound to the individual beads, and thus, the results of this
experiment produce a collection of one-bead-one-compound in a bead
pool.
[0168] In another body experiment, the individual reaction sites
are isolated after the beads distributed into the sites. The
binding of the surface molecules to the beads occurs and the
surface molecules are cleaved from the surface. The results of this
experiment produce a collection of one-bead-one-compound in a bead
pool.
[0169] In the experiment described, the individual reaction sites
are isolated after the beads distributed into the sites. The
binding of the surface molecules to the beads occurs after the
surface molecules are cleaved from the surface. The results of this
experiment produce a collection of one-bead-one-compound in a bead
pool.
[0170] The molecules synthesized or immobilized on a surface at
discrete sites. For each of the biopolymers synthesized, an anchor
moiety is incorporated. for instance, at the terminus position. The
anchor moiety is a reactive group or a group can be activated to
form a linkage to the incoming beads. After the synthesis, beads,
to which the surface molecules can adhere, are loaded onto the
surface. The surface molecules will bind to the beads upon
contacting. The surface molecules are then cleaved from the
surface. At each contact area, only one type of molecules is bound
to the individual beads, and thus, the results of this experiment
produces a collection of one-bead-one-compound in a bead pool.
[0171] In the experiment described, after the synthesis, beads, to
which the surface molecules can adhere, are loaded onto the surface
in a viscous media. The surface molecules will bind to the beads
upon contacting. The surface molecules are then cleaved from the
surface. At each contact area, only one type of molecules is bound
to the individual beads, and thus, the results of the experiment
produces a collection of one-bead-one-compound in a bead pool.
[0172] In the experiments described, the anchor moiety incorporated
in the molecules synthesized may be a biotin moiety which can bind
to streptavidin or avidin beads, a thiol group which can bind to
gold spheres, an amino group which can link to an aldehyde coated
on beads, a thiol which reacts with maleirnide on bead surface, or
other type of ligand-host systems.
[0173] The density of the surface molecules and diameters of the
beads can be adjusted so that the number of the molecules attached
to the bead can be controlled.
[0174] Tests showing the tight binding or bonding of the surface
molecules with the beads causes the immobilization of the beads.
The type of the anchor incorporated into the molecules synthesized
on surface and the acceptor of the anchor moiety on the surface of
the bead creates strong interactions so that the surface containing
synthetic molecules is essentially "sticking" to the beads.
[0175] A preferred embodiment of the present invention is the
application of the bead library generated. Following the protocol
described for "Ultrafast de novo sequencing of the human pathogen
Corynebacterium urealyticum with the Genome Sequencer System"
(www.454.com), a genomic DNA sample is obtained. Making an array of
oligos containing the required sequence, the sequences are
terminated with biotin, cleave the sequences from the array surface
with 3'OH present. Prepare a library of bead loaded oligos which
are primers designed specific for the regions of DNA sequences of
interest (DNA sequence information is available from NGBI or EBI
information service sites); mix the beads with the genomic DNA, PGR
to amplify the target DNA. This sample will be sequenced by the 454
Genome Sequencer. The target-specific sequencing will result in an
increase in the total base reads and the coverage of the unique
bases sequenced,
[0176] Various modifications and variations of the described method
and system of the invention will be apparent to those skilled in
the art without departing from the scope and spirit of the
invention. Although the invention has been described in connection
with specific preferred embodiments, it should be understood that
the invention as claimed should not be unduly limited to such
specific embodiments. Indeed, various modifications of the
described modes for carrying out the invention which are obvious to
those skilled in molecular biology, genetics, chemistry or related
fields are intended to be within the scope of the following
claims.
[0177] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it is readily apparent to those of ordinary skill
in the art in light of the teachings of this invention that certain
changes and modifications may be made thereto without departing
from the spirit or scope of the appended claims.
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